This invention is in the field of diagnostic reagents and methods for detecting deoxyribonucleic (DNA) or ribonucleic acid (RNA) sequences in test samples. The invention relates to the field of binding DNA to a solid support.
The present invention encompasses methods, reagents, compositions, and kits for detecting deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) from test samples. Embodiments of the present invention provide methods for rapid, sensitive detection of nucleic acid targets in food or clinical samples such as body fluids which contain viruses, or bacteria. These reagents and methods are adaptable to non-radioactive labeling techniques and automation and are equally applicable to other DNA and RNA containing cells such as mammalian cells and fungi.
The term "polynucleic acid probe" refers to a nucleic acid sequence having a specific binding sequence or region and one or more cytidines generally a polycytidine region wherein cytidines in the specific binding sequence are replaced with 5-methylcytidine. The specific binding sequence may be directly bindable to the nucleic acid sequence to be determined or it may be bindable to a nucleic acid sequence bound to the sequence to be determined. The specific binding sequence may be complementary to nucleic acid sequences of variable nucleic acid residues or it may be complementary to homopolymer segments such as polyadenosine, polyguanosine, polythymidine, or poly-5-methylcytidine. The polycytidine region of the polynucleic acid probe may be at the end of the probe or it may be centrally located with nucleic acid sequences on either side, for example, a polycytidine region with two specific binding sequences on either side.
The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation radioactive isotopes, enzymes, luminescent agents and dyes The term "agent" is used in a broad sense, including any molecular moiety which participates in reactions which lead to a detectable response Also biological detecting systems may be employed e.g., bacteriophage oligonucleotide conjugates.
The term "retrievable" is used in a broad sense to describe an entity which can be substantially dispersed within a medium and removed or separated from the medium by immobilization, filtering, partitioning, or the like.
The term "support" when used includes conventional supports such as nitrocellulose or nylon filters, membranes, beads, and dip sticks and microtiter plate wells, as well as retrievable magnetic supports. A solid support is a separation medium by which nucleic acid sequences can be selectively bound and then separated from other components of the reaction mixture.
Genetic information is contained in living cells in threadlike molecules of DNA. In vivo, the DNA molecule is a double helix each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T). and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
DNA consists of covalently linked chains of deoxyribonucleotides and RNA consists of covalently linked chains of ribonucleotides. The genetic code of a living organism is carried upon the DNA strand in the sequence of the base pairs.
Each nucleic acid is linked by a phosphodiester bridge between the five prime hydroxyl group of the sugar of one nucleotide and the three prime hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a 5'-hydroxyl group and another terminal end having a 3'-hydroxyl group. The terminal ends of polynucleotides are often referred to as being 5'-terminal or 3'-terminal in reference to the respective free hydroxyl group. Complementary strands of DNA and RNA form antiparallel complexes in which the 3'-terminal end of one strand is oriented and bound to the 5'-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at complementary regions. Presently, nucleic acid hybridization assays are primarily used to detect and identify unique DNA or RNA base sequences or specific genes in a complete DNA molecule, in mixtures of nucleic acid, or in mixtures of nucleic acid fragments.
The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility cancer and precancerous states, or bacterial or viral infections The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures or tissue containing bacteria may indicate the presence of antibiotic resistance, toxins, viruses, or plasmids, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease Further potential exists in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxin-producing bacteria.
One of the most widely used nucleic acid hybridization assay procedures is known as the Southern blot filter hybridization method or simply, the Southern procedure (Southern, E., J. Mol. Biol. I., 98, 503, (1975)). The Southern procedure is used to identify target DNA or RNA sequences. This procedure is generally carried out by immobilizing sample RNA or DNA to nitrocellulose sheets as the solid support. The immobilized sample RNA or DNA is contacted with radio-labeled probe strands of DNA having a base sequence complementary to the target sequence carrying a radioactive moiety which can be detected. Hybridization between the probe and the sample DNA is allowed to take place.
The hybridization process is generally very specific. The labeled probe will not combine with sample DNA or RNA if the two nucleotide entities do not share substantial complementary base pair organization. Hybridization can take from three to 48 hours depending on given conditions.
However, as a practical matter there is always non-specific binding of the labeled probe to supports which appears as "background noise" on detection. Background noise reduces the sensitivity of an assay. Unhybridized DNA probe is subsequently washed away. The nitrocellulose sheet is placed on a sheet of X-ray film and allowed to expose. The X-ray film is developed with the exposed areas of the film identifying DNA fragments which have been hybridized to the DNA probe and therefore have the base pair sequence of interest.
The use of radioactive labeling agents in conjunction with Southern assay techniques have allowed the application of nucleic acid assays to clinical samples. Radioactive decay is detectable even in clinical samples containing extraneous proteinaceous and organic material. However, the presence of extraneous proteinaceous and organic material may contribute to nonspecific binding of the probe to the solid support. Moreover, the use of radioactive labeling techniques requires a long exposure time to visualize bands on X-ray film A typical Southern procedure may require 1 to 7 days for exposure. The use of radioactive labeling agents further requires special laboratory procedures and licenses.
The above problems associated with assays involving radioisotopic labels have led to the development of techniques employing nonisotopic labels. Examples of nonisotopic labels include enzymes, luminescent agents, dyes and biological detecting systems such as bacteriophage. Luminescent labels emit light upon excitation by an external energy source and may be grouped into categories dependent upon the source of the exciting energy, including: radioluminescent labels deriving energy from high energy particles; chemiluminescent labels which obtain energy from chemical reactions; bioluminescent labels wherein the exciting energy is applied in a biological system; and photoluminescent or fluorescent labels which are excitable by units of electromagnetic radiation (photons) of infrared, visual or ultraviolet light See, generally, Smith et al., Ann. Clin. Biochem., 18:253, 274 (1981).
Nonisotopic assay techniques employing labels excitable by nonradioactive energy sources avoid the health hazards and licensing problems encountered with radioisotopic label assay techniques. Moreover, nonisotopic assay techniques hold promise for rapid detection avoiding the long exposure time associated with the use of X-ray film.
However, nonisotopic assays have not conveyed the sensitivity or specificity to assay procedures necessary to be considered reliable. In luminescent assays, the presence of proteins and other molecules carried in biological samples may cause scattering of the exciting light or may absorb light in the spectrum of emission of the luminescent label, resulting in a quenching of the luminescent probe.
In enzymatic assays, the presence of proteins and other molecules carried in biological samples may interfere with the activity of the enzyme.
Similarly, in colorimetric assays, the change in color may not be detectable over proteins and other materials carried in biological samples.
The use of polynucleic acid probes in diagnostic assays for DNA and RNA is extensively taught in the prior art.
U.S. Pat. No. 4,358,535 extensively discusses the detection of various pathogens using labeled polynucleotide probes. Thus probes, hybridization conditions and methods for detecting labeled probes are well known.
U.S. Pat. No. 4,689,295 describes a test for salmonella using probes specific for salmonella DNA This patent describes in detail the culturing, lysing, and sample preparation techniques. This patent also describes fixing DNA to nitrocellulose filters.
U.S. Pat. No. 4,626,501, EPA 259,186 and EPA 251,527 describe DNA probe assays on solid supports.
PCT/US 86/01280 application describes various configurations of nucleic acid hybridization assays widely applicable to detecting nucleic acid sequences specific to various bacteria and viruses.
U.S. Pat. No. 4,139,346 describes nucleic acid probes covalently bound to a support paper modified with aminobenzyloxymethyl groups.
U.K. Patent Application GB 2169403A describes the formation of a complex where the DNA or RNA to be detected is bound to two probes in different complementary regions. One probe is labeled with a detectable marker and the other probe is bindable on a support so that it can be captured by a membrane having a binding partner.
The prior art also describes bisulfite catalyzed transamination reactions with cytidine.
Of the four nucleic acids, cytidine uniquely undergoes transamination reaction in the presence of bisulfite.
Bisulfite modifications of nucleic acids is extensively discussed in Prog. Nucl. Acid Res. Mol. Biol., 16: 75 (1976). "Bisulfite Modification of Nucleic Acids and their Constituents," (Hikoya Hayatsu).
Nucleic Acid Research, Volume 12, Number 2, 1984, pg. 989, describes the attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite catalyzed transamination reaction.
Biochemical and Biophysical Research Communications, Vol 142, No. 2, 1987, Jan. 30, 1987, pp. 519, describes the linking of cytidine to a biotin hydrazide using a bisulfite catalyzed reaction.
Analytical Biochemistry, 157, 199-207 (1986), pp 199, describes N.sup.4 (6-aminohexyl)cytidine and cytidine-containing nucleotides where these compounds are formed using a bisulfite catalyzed transamination reactions.
Journal of the American Chemical Society, 95, 14, July 14, 1973, pp. 4746, describes bisulfite catalyzed isotope exchange on cytidine.
Nucleic Acids Research, Vol. 9, Nov. 5, 1981, pp. 1203 coupling t-RNA with N-hydroxysuccinimide by way of a bisulfite catalyzed transamination.
J. Chem. Biol., 1979, 78(i), 61-75 describes the bisulfite catalyzed transamination of cytidine and acylhydrazides.